Many drugs currently on the market were developed from leads identified from high throughput screening (HTS). Targets of therapeutic interest used in HTS are often recombinant proteins produced from cloned genes which can be expressed in different ways. A large compound collection is typically screened against these proteins for the identification of inhibitors.
During the last ten years the size of the proprietary compound collection has increased exponentially as a result of systematic application of combinatorial chemistry to different projects. Combinatorial chemistry nowadays generates large compound libraries that complement other compound libraries available from traditional medicinal chemistry and natural sources. The development and application of robotics and automation have made it feasible to test large numbers of compounds in a short period of time. Several new detection systems are used for the identification of potential lead molecules.
Recently, nuclear magnetic resonance (NMR) has emerged as a powerful method for the detection of small molecules that interact with targets of pharmaceutical interest. Although NMR is not a sensitive technique, it has the advantage that it is less subject to artifacts observed with other systems of detection. Recent developments in cryogenic NMR probe technology have reduced the period of time or the amount of protein necessary for the screening.
NMR methods have been used for screening a large compound collection against isotopically labeled proteins. Chemical shift changes of cross peaks in a 15N—1H HSQC spectrum of the target protein are monitored in the presence of a compound mixture. Deconvolution of the mixture then results in the identification of the molecule interacting with the protein (i.e., the compound responsible for the chemical shift changes). When the three dimensional structure of the protein is known and the sequence specific NMR assignments of the protein backbone resonances have been obtained, the method provides important structural information of the ligand binding site and ligand binding mode.
Another method for performing NMR screening is based on the detection of the ligand resonances. Several NMR parameters have been proposed in the literature as a tool for ligand identification. These methodologies permit rapid deconvolution of the screened mixtures and are particularly suited for the identification of medium to low affinity ligands.
However, these techniques suffer from some drawbacks. First, no structural information regarding the binding site is directly available. Second, high affinity ligands and molecules that bind covalently to the receptor escape detection because of the large excess of the test compound over protein typically used in these experiments. That is, compounds interacting tighter to the protein or compounds that have a slow on-rate will not be detected because the residence time of these compounds within the protein is longer than the window of the mixing time (e.g., 1 to 2 seconds) employed in the NMR experiments. Third, compounds with poor solubilities that are potential ligands are difficult to detect since the method requires the observation of the ligand signals.
Thus, what is needed are additional NMR methods that can be used to detect ligands to target molecules, such as proteins, without the drawbacks associated with typical ligand-observed screening experiments.